This SPIRAT proposal aims to develop and clinically evaluate gene therapy for HIV infection. Project 1 will focus on the development and/or comparison of murine retrovirus, HIV, AAV and HIV/AAV chimeric vectors for delivery of ribozymes and other anti-HIV genes into primary hematopoietic cells, including progenitor cells. In contrast to MLV, AAV and HIV are able to infect non-dividing cells (e.g. monocyte/macrophages). HIV and HIV/AAV vectors have the further advantage of being activated and rescued by HIV infection, as well as tracking their RNA transcripts with HIV NA intracellularly. Through close interaction with the other projects, we hope to develop a gene therapy strategy that can realistically confer therapeutic benefits and that can be ready for clinical evaluation within the term of this SPIRAT award. The specific aims are: (1) Development of AAV vector for gene delivery and assessment of efficacy-Efforts will be directed at achieving sufficiently high titers of virus vector for efficient transduction of primary human cells, including unstimulated human PBL, terminally differentiated macrophage/monocytes and CD34+ stem cells in the absence and presence of growth factors. The transduced cells will be assessed for the degree of protection from challenge by HIV infection. (2) Development of HIV vectors for gene delivery-Efforts will include construction of packaging cell lines derived from the dual tropic viruses HIV-1(MN) and HIV-2(KR) and construction of HIV-2 vectors. An HIV-2 vector would be impervious to HIV-1 specific interventions such as HIV-1 targeted antisense RNA and ribozymes, but still be rescuable by HIV- 1. The efficiency of transduction of primary PBL by vectors delivering ribozymes or other anti-HIV genes will be determined. (3) Development of HIV/AAV chimeric vector - an HIV-2 vector will be subcloned into an AAV vector containing the ITR sequences, and be delivered as an AAV vector. Such a vector has the combined advantages of AAV and HIV: Like AAV, it can be packaged to relatively high titers with AAV proteins and can infect CD34+ cells. However, the integrated vector can be activated and rescued upon HIV infection. Furthermore, RNA transcripts from this vector (e.g. ribozyme) is likely to track with HIV RNA in infected cells. (4) Comparison of gene delivery by AAV vs. retroviral vectors into T-cell lines, human PBL, human macrophages/monocytes and CD34+ stem cells.